Method for the Treatment of Multiple Sclerosis by Inhibiting Il-17 Activity

ABSTRACT

The present invention provides a method for the treatment and/or prophylaxis of multiple sclerosis (MS) comprising administering a therapeutically effective amount of an inhibitor of IL-17 activity.

The present invention relates generally to methods of treating multiplesclerosis and more specifically to the use of inhibitors of IL-17activity for the manufacture of a medicament for the treatment ofmultiple sclerosis.

Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, is apro-inflammatory cytokine which stimulates the secretion of a wide rangeof other cytokines from various non-immune cells. IL-17 is capable ofinducing the secretion of IL-6, IL-8, PGE2, MCP-1 and G-CSF by adherentcells like fibroblasts, keratinocytes, epithelial and endothelial cellsand is also able to induce ICAM-1 surface expression, proliferation of Tcells, and growth and differentiation of CD34+human progenitors intoneutrophils when cocultured in presence of irradiated fibroblasts(Fossiez et al., 1998, Int. Rev. Immunol. 16, 541-551). IL-17 ispredominantly produced by activated memory T cells and acts by bindingto a ubiquitously distributed cell surface receptor (IL-17R) (Yao etal., 1997, Cytokine, 9, 794-800). A number of homologues of IL-17 havebeen identified which have both similar and distinct roles in regulatinginflammatory responses. For a review of IL-17 cytokine/receptor familiessee Dumont, 2003, Expert Opin. Ther. Patents, 13, 287-303.

IL-17 may contribute to a number of diseases mediated by abnormal immuneresponses, such as rheumatoid arthritis and air-way inflammation, aswell as organ transplant rejection and antitumour immunity. Inhibitorsof IL-17 activity are well known in the art, for example an IL-17R:Fcfusion protein was used to demonstrate the role of IL-17 incollagen-induced arthritis (Lubberts et al., J. Immunol. 2001,167,1004-1013) and neutralising polyclonal antibodies have been used toreduce peritoneal adhesion formation (Chung et al., 2002, J. Exp. Med.,195, 1471-1478). Neutralising monoclonal antibodies are commerciallyavailable (R&D Systems UK).

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinatingdisease of the central nervous system (CNS), which is believed to resultfrom a coordinated autoimmune attack against myelin antigens. There isconsiderable clinical and pathological heterogeneity in MS patients andthe sequence of events that initiate the disease remain largely unknown.The clinical progression of MS may be largely attributed to threedisease processes; inflammation, demyelination and axonalloss/neurodegeneration.

Immune mediated inflammatory lesions within the CNS are thought toresult primarily from an infiltration of autoreactive CD4⁺ lymphocytes(Th1) which recognise myelin proteins presented on MHC class IImolecules by antigen presenting cells. This interaction causesstimulation of Th1 cells which release proinflammatory cytokines (mainlyTNF-α & IFN-γ) resulting in proliferation of T-cells, activation ofB-cells and macrophages, upregulation of adhesion molecules anddisruption of the blood-brain barrier. Such events ultimately lead toloss of oligodendrocytes & axons and the formation of a demyelinatedplaque. This is the hallmark of MS and consists of a demarcated lesionwhere myelin sheaths are completely lost and demyelinated axons areembedded in glial scar tissue. Demyelination may also occur as aconsequence of specific recognition and opsonization of myelin antigensby autoantibodies. The most important target antigen is suggested to bemyelin oligodendrocyte protein (MOG), which is present on the surface ofthe myelin sheath. Destruction of antibody-opsonized myelin is thenaccomplished either by complement or activated macrophages. Axonal lossand neurodegeneration subsequent to inflammation are thought to beresponsible for the accumulation of irreversible neurologicalimpairment, characteristic of secondary progressive MS.

The clinical features of MS vary from headaches and blurred vision tosevere ataxia, blindness and paralysis. MS affects all ages but firstsymptoms generally occur between 18 and 50 years and disease durationhas been estimated at >25 years with a significant proportion ofpatients dying from causes unrelated to MS. In the majority of patients(˜80%) the disease takes a relapsing-remitting (RR-MS) course withexacerbation of symptoms, which is rapid in onset (hours to days)followed by a slower recovery. The frequency and duration of relapsesare unpredictable but average 1.5 per year and can be followed bycomplete recovery. With time, recovery from relapses may not be completeand a gradual worsening of disease occurs. This worsening of disease isindependent of relapse rate and is classified as secondary progressiveMS (SP-MS), accounting for approximately 10% of MS patients. Theremaining 10% of MS patients have a primary progressive (PP-MS) coursewhere disability worsens at a steadv rate from onset of the disease.

Currently licensed therapies are the beta-interferons; Interferonbeta-1b (Betaseron; Berlex), Interferon beta-1a (Avonex; Biogen, Rebif;Serono) and glatimer acetate (Copaxone; Teva). These agents have beenshown to reduce relapse rate during the relapsing-remitting phase of thedisease in approximately 30% of patients. There is currently no methodavailable for identifying the responder population before therapy.Intravenous steroids (prednisolone is most commonly used) are used tohasten remission after relapse but do not have long term efficacy. Theanti-cancer agent, mitoxantrone (Novantrone), is approved as animmunosuppressant in progressive-relapsing and secondary-progressivepatients, but its use and dose is limited by cardiotoxicity. In Europeazathioprine has also been used as an immunosuppressant.

Prescribing decisions seem to be driven by evidence-based medicine and arecent report by the American Association of Neurologists (Goodin D S etal; Neurology Jan. 22, 2002;58(2):169-78) is a key document. Theconsensus amongst many neurologists is that early, aggressive therapywith beta-interferons was desirable in increasing the time to firstrelapse and limiting the overall disease load, although it wasrecognised that there was no evidence that this approach showedlong-term benefit on EDSS score (a measure of disease-relateddisability). Beta-interferons were seen as sub-optimal therapy andglatirimer acetate as having a different mechanism of action, which mayallow it to be used (alone or in combination) in patients that do notrespond to interferons. Individualised therapy based on mechanistic(MRI, genetic, neurological) markers of disease was seen as a worthwhilegoal, as were therapies with a novel mechanism of action. There iscurrently no satisfactory diagnostic marker for multiple sclerosis.

There is a clear need for disease modifying therapies. Agents withdifferent mechanisms of action are needed and may allow therapy to betailored to different stages of the disease. An orally active agent isyet to be licensed in the relapsing-remitting form of the disease andthis would represent a clear improvement over current therapy ifsignificant efficacy was associated with the mechanism. Furthermore,there is a clear requirement for therapies that show efficacy in theprimary or secondary progressive phases of the disease and have areasonable side-effect profile.

Whether IL-17 plays any kind of role in the pathogenesis of MS isunknown. Microarray analysis of MS lesions obtained at autopsy haverevealed increased transcripts of many different genes encodinginflammatory cytokines, including, IL-17 (Lock et al., 2002, NatureMedicine, 8, 500-508). An increased number of IL-17 expressingmononuclear cells have been detected in blood and cerebrospinal fluidfrom patients with MS (Matusevicius et al., 1999, Multiple Sclerosis, 5,101-104) but as the authors point out cytokine mRNA expression is notnecessarily identical to cytokine protein production.

Surprisingly we have been able to demonstrate that inhibitors of IL-17activity are active in an animal model of MS. Specifically we have beenable to demonstrate that an anti-IL-17 antibody that inhibits IL-17activity is active in animal models of MS. Hence, the present inventionprovides a method for the treatment and/or prophylaxis of MS comprisingadministering a therapeutically effective amount of an inhibitor ofIL-17 activity. The invention also provides the use of an inhibitor ofIL-17 activity for the manufacture of a medicament for the treatmentand/or prophylaxis of multiple sclerosis.

The term ‘IL-17 activity’ as used herein refers to the spectrum ofactivity understood in the art for IL-17 for example, the induction ofsecretion of IL-6 or IL-8 from fibroblasts by IL-17 (Yao et al., 1995,Journal of Immunology, 155,5483-5486).

An inhibitor of IL-17 activity according to the present invention is anagent that interferes with the activity of IL-17, in particular theactivity of IL-17 in MS. Particularly preferred are agents whichinterfere with the activity of IL-17 in MS in humans. Inhibitorsaccording to the present invention may partially or completely inhibitIL-17 activity. Inhibitors of use in the present invention includewithout limitation, inhibitors that are capable of interacting with(e.g. binding to, or recognising) IL-17 or the IL-17 receptor (L17 R) ora nucleic acid molecule encoding IL-17 or IL-17R, or are capable ofinhibiting the expression of IL-17 or IL-17R or are capable ofinhibiting the interaction between IL-17 and IL-17R. Such inhibitors maybe, without limitation, antibodies, nucleic acids (e.g. DNA, RNA,antisense RNA and siRNA), carbohydrates, lipids, proteins, polypeptides,peptides, peptidomimetics, small molecules and other drugs.

Examples of suitable inhibitors include, but are not limited to, asynthetic functional fragment of the IL-17 receptor that binds to IL-17and interferes with binding to the native IL-17 receptor, an antibodythat binds to IL-17 or to the IL-17 receptor and interferes with IL-17receptor-ligand interaction, an antisense nucleic acid molecule thatspecifically hybridizes to mRNA encoding IL-17 or the IL-17 receptor ora small molecule or other drug which inhibits the activity of IL-17 orits receptor.

Inhibitors of IL-17 activity are well known in the art as are methods ofidentifying and producing such inhibitors. Examples include, IL-17R:Fcfusion proteins (Lubberts et al., J. Immunol. 2001,167, 1004-1013) andneutralising antibodies (Chung et al., 2002, J. Exp. Med., 195,1471-1478; Ferretti, 2003, Journal of Immunology, 170, 2106-2112).Agents that may be suitable inhibitors can be selected from a widevariety of candidate agents. Examples of candidate agents include butare not limited to, nucleic acids (e.g. DNA and RNA), carbohydrates,lipids, proteins, polypeptides, peptides, peptidomimetics, smallmolecules and other drugs. Agents can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is suited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145;U.S. Pat. No. 5,738,996; and U.S. Pat. No. 5,807,683).

Examples of suitable methods based on the present description for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al.,1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al., 1994, J.Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carrell et al.,1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew.Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem.37:1233.

Libraries of compounds may be presented, for example, in solution (e.g.Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991,Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria(U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484;and 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA89:1865-1869) or phage (Scott and Smith, 1990, Science 249:386-390;Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol. Biol.222:301-310).

In one example, the inhibitor for use in the present invention may be anucleic acid. In particular IL-17 or IL-17R nucleic acid molecules maybeused as anti-sense molecules, to alter the expression of theirrespective polypeptides by binding to complementary nucleic acids. IL-17or IL-17R nucleic acids may be obtained using standard cloningtechniques from for example genomic DNA or cDNA or can be synthesisedusing well known and commercially available techniques. The IL-17 orIL-17R nucleic acids may contain one or more nucleotide substitutions,additions or deletions into the nucleotide sequence of an IL-17 orIL-17R nucleic acid. Standard techniques known to those of skill in theart can be used to introduce mutations, including, for example,site-directed mutagenesis and PCR-mediated mutagenesis. An antisensenucleic acid according to the present invention includes a IL-17 orIL-17R nucleic acid capable of hybridising by virtue of some sequencecomplementarity to a portion of an RNA (preferably mRNA) encoding therespective polypeptide. The antisense nucleic acid can be complementaryto a coding and/or non-coding region of an mRNA encoding such apolypeptide. Most preferably, the antisense nucleic acids result ininhibition of the expression of the IL-17 or IL-17R polypeptide. Thus,the present invention provides a method for the treatment and/orprophylaxis of MS comprising administering a therapeutically effectiveamount of an inhibitor of IL-17 activity wherein the inhibitor comprisesat least eight nucleotides that are antisense to a gene or cDNA encodinga IL-17 or IL-17R polypeptide. The invention also provides the use ofnucleic acids comprising at least eight nucleotides that are antisenseto a gene or cDNA encoding a IL-17 or IL-17R polypeptide for themanufacture of a medicament for the treatment and/or prophylaxis of MS.

Most preferably, an inhibitor for use in the treatment and/orprophylaxis of MS is an antibody that interacts with (i.e. binds to orrecognises) IL-17 or its receptor and inhibits the activity of IL-17.Accordingly, there is provided the use of an antibody that inhibits theactivity of IL-17 for the manufacture of a medicament for the treatmentand/or prophylaxis of MS. Also provided is a method of treatment and/orprophylaxis of MS in a subject comprising administering to said subjecta therapeutically effective amount of an antibody that inhibits theactivity of IL-17.

In one example the antibodies selectively interact with IL-17.Selectively interacting with (e.g. recognising or binding to) means thatthe antibodies have a greater affinity for IL-17 polypeptides than forother polypeptides. Examples of suitable antibodies are those thatinhibit the activity of IL-17 by binding to IL-17 in such a manner as toprevent it being biologically active, for example by preventing thebinding of IL-17 to its receptor. Accordingly, there is provided by thepresent invention the use of an anti-IL-17 antibody for the manufactureof a medicament for the treatment and/or prophylaxis of MS. Alsoprovided is a method of treatment and/or prophylaxis of MS in a subjectcomprising administering to said subject a therapeutically effectiveamount of an anti-IL-17 antibody.

In another example the antibodies selectively interact with the IL-17receptor. Selectively interacting with (e.g. recognising or binding to)means that the antibodies have a greater affinity for the IL-17 receptorpolypeptide than for other polypeptides. Examples of suitable antibodiesare those that inhibit the activity of IL-17 by preventing IL-17mediated signalling from the receptor, for example by preventing IL-17from binding to the IL-17 receptor. Accordingly, there is provided bythe present invention the use of an anti-IL-7R antibody for themanufacture of a medicament for the treatment and/or prophylaxis of MS.Also provided is a method of treatment and/or prophylaxis of MS in asubject comprising administering to said subject a therapeuticallyeffective amount of an anti-IL-17R antibody.

IL-17 or IL-17 receptor polypeptides or cells expressing saidpolypeptides can be used to produce antibodies which specificallyrecognise said polypeptides. The IL-17 and IL-17R polypeptides may be‘mature’ polypeptides or biologically active fragments or derivativesthereof. IL-17 and IL-17R polypeptides may be prepared by processes wellknown in the art from genetically engineered host cells comprisingexpression systems or they may be recovered from natural biologicalsources. In the present application, the term “polypeptides” includespeptides, polypeptides and proteins. These are used interchangeablyunless otherwise specified. IL-17 or IL-17R polypeptides may in someinstances be part of a larger protein such as a fusion protein forexample fused to an affinity tag. Antibodies generated against thesepolypeptides may be obtained by administering the polypeptides to ananimal, preferably a non-human animal, using well-known and routineprotocols, see for example Handbook of Experimental Immunology, D. M.Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England,1986. Many warm-blooded animals, such as rabbits, mice, rats, sheep,chickens, cows or pigs may be immunised. However, mice, rabbits, pigsand rats are generally preferred.

Anti-IL-17 and anti-IL-17 receptor antibodies for use in the presentinvention include whole antibodies and functionally active fragments orderivatives thereof and may be, but are not limited to, polyclonal,monoclonal, multi-valent, multi-specific, humanized or chimericantibodies, single chain antibodies, Fab fragments, Fab′ and F(ab′)₂fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Antibodies include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.molecules that contain an antigen binding site that specifically bindsan antigen. The immunoglobulin molecules of the invention can be of anyclass (e.g. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulinmolecule.

Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, 1975, Nature,256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies for use in the invention may also be generated using singlelymphocyte antibody methods by cloning and expressing immunoglobulinvariable region cDNAs generated from single lymphocytes selected for theproduction of specific antibodies by for example the methods describedby Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-7848and in WO92/02551.

Humanized antibodies are antibody molecules from non-human specieshaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework region from a human immunoglobulinmolecule (see, e.g. U.S. Pat. No. 5,585,089).

Chimeric antibodies are those antibodies encoded by immunoglobulin genesthat have been genetically engineered so that the light and heavy chaingenes are composed of immunoglobulin gene segments belonging todifferent species. These chimeric antibodies are likely to be lessantigenic. Bivalent antibodies may be made by methods known in the art(Milstein et al., 1983, Nature 305:537-539; WO 93/08829, Traunecker etal., 1991, EMBO J. 10:3655-3659). Multi-valent antibodies may comprisemultiple specificities or may be monospecific (see for example WO92/22853).

The antibodies for use in the present invention can also be generatedusing various phage display methods known in the art and include thosedisclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50),Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough etal. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 1879-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778 can also be adapted to producesingle chain antibodies to IL-17 or IL-17R polypeptides. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

Antibody fragments and methods of producing them are well known in theart, see for example Verma et al., 1998, Journal of ImmunologicalMethods, 216, 165-181.

Particular examples of antibody fragments for use in the presentinvention are Fab′ fragments which possess a native or a modified hingeregion. A number of modified hinge regions have already been described,for example, in U.S. Pat. No. 5,677,425, WO9915549, and WO9825971 andthese are incorporated herein by reference

Further examples of particular antibody fragments for use in the presentinvention include those described in International patent applicationsPCT/GB2004/002810, PCT/GB2004/002870 and PCT/GB2004/002871 (all filed on1 Jul. 2004). In particular the modified antibody Fab fragmentsdescribed in International patent application PCT/GB2004/002810 arepreferred. These Fab fragments comprise a heavy and light chain pair,V_(H)/C_(H)1 and V_(L)/C_(L) covalently linked through interchaincysteines in the heavy and light chain constant regions and arecharacterised in that the heavy chain constant region terminates at theinterchain cysteine of C_(H)1. The term ‘interchain cysteine’ refers toa cysteine in the heavy or light chain constant region that would bedisulphide linked to a cysteine in the corresponding heavy or lightchain constant region encoded in a naturally occurring germline antibodygene. In particular the interchain cysteines are a cysteine in theconstant region of the light chain (C_(L)) and a cysteine in the firstconstant region of the heavy chain (C_(H)1) that are disulphide linkedto each other in naturally occurring antibodies. Examples of suchcysteines may typically be found at position 214 of the light chain andposition 233 of the heavy chain of human IgG1, position 127 of the heavychain of human IgM, IgE, IgG2, IgG3, IgG4 and position 128 of the heavychain of human IgD and IgA2B, as defined by Kabat et al., 1987, inSequences of Proteins of Immunological Interest, US Department of Healthand Human Services, NIH, USA. In murine IgG, interchain cysteines may befound at position 214 of the light chain and position 235 of the heavychain. It will be appreciated that the exact positions of thesecysteines may vary from that of naturally occurring antibodies if anymodifications, such as deletions, insertions and/or substitutions havebeen made to the antibody Fab fragment. These antibody Fab fragments maybe prepared by any suitable method known in the art. For example, theantibody Fab fragment may be obtained from any whole antibody,especially a whole monoclonal antibody, using any suitable enzymaticcleavage and/or digestion techniques, for example by treatment withpepsin or papain and c-terminal proteases. Preferably these antibody Fabfragments are prepared by the use of recombinant DNA techniquesinvolving the manipulation and re-expression of DNA encoding antibodyvariable and constant regions. Standard molecular biology techniques maybe used to modify, add or delete further amino acids or domains asdesired. Any alterations to the variable or constant regions are stillencompassed by the terms ‘variable’ and ‘constant’ regions as usedherein. Preferably PCR is used to introduce a stop codon immediatelyfollowing the codon encoding the interchain cysteine of C_(H)1, suchthat translation of the C_(H)1 domain stops at the interchain cysteine.Methods for designing suitable PCR primers are well known in the art andthe sequences of antibody C_(H)1 domains are readily available (Kabat etal., supra). Alternatively stop codons may be introduced usingsite-directed mutagenesis techniques such as those described in White(Ed.), PCR Protocols: Current Methods and Applications (1993). In oneexample the constant regions in these fragments are derived from IgG1and the interchain cysteine of C_(L) is at position 214 of the lightchain and the interchain cysteine of C_(H)1 is at position 233 of theheavy chain. Examples of human and murine constant region sequences foruse in these fragments are provided in SEQ ID Nos 1-4 and FIG. 13; humanheavy chain constant region C_(H)1 which terminates at the interchaincysteine (SEQ ID NO:1); human light chain constant region (SEQ ID NO:2);murine heavy chain constant region C_(H)1 which terminates at theinterchain cysteine (SEQ ID NO:3); murine light chain constant region(SEQ ID NO:4).

If desired an antibody for use in the present invention may beconjugated to one or more effector molecule(s). The term effectormolecule as used herein includes, for example, antineoplastic agents,drugs, toxins, biologically active proteins, for example enzymes, otherantibody or antibody fragments, synthetic or naturally occurringpolymers, nucleic acids and fragments thereof e.g. DNA, RNA andfragments thereof, radionuclides, particularly radioiodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy. In one example, anti-IL-17 or anti IL-17R antibodiescan be conjugated to an effector molecule, such as a cytotoxic agent, aradionuclide or drug moiety to modify a given biological response. Forexample, the therapeutic agent may be a drug moiety which may be aprotein or polypeptide possessing a desired biological activity. Suchmoieties may include, for example and without limitation, a toxin suchas abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a proteinsuch as tumour necrosis factor, α-interferon, β-interferon, nerve growthfactor, platelet derived growth factor or tissue plasminogen activator,a thrombotic agent or an anti-angiogenic agent, e.g. angiostatin orendostatin, or, a biological response modifier such as a lymphokine,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), nerve growth factor (NGF) or othergrowth factor.

In another example the effector molecules may be cytotoxins or cytotoxicagents including any agent that is detrimental to (e.g. kills) cells.Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Effector molecules alsoinclude, but are not limited to, antimetabolites (e.g. methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g. mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g. daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin),bleomycin, mithramycin, anthramycin (AMC), calicheamicins orduocarmycins), and anti-mitotic agents (e.g. vincristine andvinblastine).

Other effector molecules may include radionuclides such as ¹¹¹In and⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Iridium¹⁹² andTungsten¹⁸⁸/Rhenium¹⁸⁸; or drugs such as but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.Techniques for conjugating such effector molecules to antibodies arewell known in the art (see, Hellstrom et al., Controlled Drug Delivery,2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982,Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology andTherapeutics, 83, 67-123). In one example, the antibody or fragmentthereof is fused via a covalent bond (e.g. a peptide bond), atoptionally the N-terminus or the C-terminus, to an amino acid sequenceof another protein (or portion thereof; preferably at least a 10, 20 or50 amino acid portion of the protein). Preferably the antibody, orfragment thereof, is linked to the other protein at the N-terminus ofthe constant domain of the antibody. Recombinant DNA procedures may beused to create such fusions, for example as described in WO 86/01533 andEP 0392745.

In another example the effector molecule may increase half-life in vivo,and/or decrease immunogenicity and/or enhance the delivery of anantibody across an epithelial barrier to the immune system. Examples ofsuitable effector molecules include polymers and proteins such asalbumin and albumin binding proteins. Examples of suitable polymersinclude any synthetic or naturally occurring substantiallywater-soluble, substantially non-antigenic polymer including, forexample, optionally substituted straight or branched chain polyalkylene,polyalkenylene, or polyoxyalkylene polymers or branched or unbranchedpolysaccharides, e.g. a homo- or hetero-polysaccharide such as lactose,amylose, dextran or glycogen. Particular optional substituents which maybe present on the above-mentioned synthetic polymers include one or morehydroxy, methyl or methoxy groups. Particular examples of syntheticpolymers include optionally substituted straight or branched chainpoly(ethyleneglycol), poly(propyleneglycol), poly(vinylalcohol) orderivatives thereof, especially optionally substitutedpoly(ethyleneglycol) such as methoxypoly(ethyleneglycol). Preferably thepolymer is a polyalkylene oxide such as polyethylene glycol (PEG).

In one example antibodies for use in the present invention are attachedto poly(ethyleneglycol) (PEG) moieties. In one particular example theantibody is an antibody fragment and the PEG molecules may be attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids mayoccur naturally in the antibody fragment or may be engineered into thefragment using recombinant DNA methods. See for example U.S. Pat. No.5,219,996. Multiple sites can be used to attach two or more PEGmolecules. Preferably PEG molecules are covalently linked through athiol group of at least one cysteine residue located in the antibodyfragment. Where a thiol group is used as the point of attachmentappropriately activated effector molecules, for example thiol selectivederivatives such as maleimides and cysteine derivatives may be used.

Preferably, the antibody is a modified Fab fragment, such as a Fab′which is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalentlyattached thereto, e.g. according to the method disclosed in EP 0948544[see also “Poly(ethyleneglycol) Chemistry, Biotechnical and BiomedicalApplications”, 1992, J. Milton Harris (ed), Plenum Press, New York,“Poly(ethyleneglycol) Chemistry and Biological Applications”, 1997, J.Milton Harris and S. Zalipsky (eds), American Chemical Society,Washington D.C. and “Bioconjugation Protein Coupling Techniques for theBiomedical Sciences”, 1998, M. Aslam and A. Dent, Grove Publishers, NewYork; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002,54:531-545]. The total amount of PEG attached to the fragment may bevaried as desired, but will generally be in an average molecular weightrange from 250 to 100,000Da, preferably from 5,000 to 50,000Da, morepreferably from 10,000 to 40,000Da and still more preferably from 20,000to 40,000Da The size of PEG may in particular be selected on the basisof the intended use of the product, for example ability to localize tocertain tissues such as tumors or extend circulating half-life (forreview see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545).

In one embodiment PEG is attached to a cysteine in the hinge region of aFab′. In one example, a PEG modified Fab′ fragment has a maleimide groupcovalently linked to a single thiol group in a modified hinge region. Alysine residue may be covalently linked to the maleimide group and toeach of the amine groups on the lysine residue may be attached amethoxypoly(ethyleneglycol)polymer having a molecular weight ofapproximately 20,000 Da. The total molecular weight of the PEG attachedto the Fab′ fragment may therefore be approximately 40,000 Da.

In another preferred embodiment an antibody fragment for use in thepresent invention is a PEGylated (i.e. has PEG (poly(ethyleneglycol))covalently attached thereto) Fab fragment as described in InternationalApplication Number PCT/GB2004/002810 (filed on 1 Jul. 2004). ThisPEGylated Fab fragment is a Fab fragment in which the heavy chainterminates at the interchain cysteine of C_(H)1 and the PEG attached tothe fragment, preferably PEG-maleimide, is covalently linked to theinterchain cysteine of C_(L) and the interchain cysteine of C_(H)1.Preferably the interchain cysteine of C_(L) is at position 214 of thelight chain and the interchain cysteine of C_(H)1 is at position 233 ofthe heavy chain. As discussed above the total amount of PEG attached tothe fragment may be varied as desired. In one example each polymerattached to the Fab preferably has a molecular weight of approximately20,000 Da. For example, the molecular weight may be 15,000-25,000Da, orpreferably 18,000-22,000Da, and even more preferably 19,000-21,000Da.The total molecular weight of the PEG attached to the antibody istherefore approximately 40,000 Da.

PEG is attached to these fragments by first reducing the interchaindisulphide bond between the interchain cysteines of C_(L) and C_(H)1 andsubsequently attaching the PEG to the free thiols. Once PEG is attachedto the interchain cysteines there is no interchain disulphide linkagebetween the heavy and light chain. Suitable reducing agents for reducingthe interchain disulphide bond are widely known in the art for examplethose described in Singh et al., 1995, Methods in Enzymology, 251,167-73. Particular examples include thiol based reducing agents such asreduced glutathione (GSH), β-mercaptoethanol (β-ME),β-mercaptoethylamine (β-MA) and dithiothreitol (DTT). Other methodsinclude using electrolytic methods, such as the method described inLeach et al., 1965, Div. Protein. Chem, 4, 23-27 and usingphotoreduction method such as the method described in Ellison et al.,2000, Biotechniques, 28 (2), 324-326. Preferably however, the reducingagent is a non-thiol based reducing agent, preferably one of thetrialkylphosphine reducing agents (Ruegg U T and Rudinger, J., 1977,Methods in Enzymology, 47, 111-126; Burns J et al., 1991, J. Org. Chem,56, 2648-2650; Getz et al., 1999, Analytical Biochemistry, 273, 73-80;Han and Han, 1994, Analytical Biochemistry, 220, 5-10; Seitz et al.,1999, Euro. J. Nuclear Medicine, 26, 1265-1273), particular examples ofwhich include tris(2-carboxyethyl)phosphine (TCEP), tris butyl phosphine(TBP), tris-(2-cyanoethyl) phosphine, tris-(3-hydroxypropyl)phosphine(THP) and tris-(2-hydroxyethyl)phosphine. Most preferred are thereducing agents TCEP and THP. It will be clear to a person skilled inthe art that the concentration of reducing agent can be determinedempirically, for example, by varying the concentration of reducing agentand measuring the number of free thiols produced. Typically the reducingagent is used in excess over the antibody fragment for example between 2and 1000 fold molar excess. Preferably the reducing agent is in 2, 3, 4,5, 10, 100 or 1000 fold excess. In one embodiment the reductant is usedat between 2 and 5 mM.

The reduction and PEGylation reactions may generally be performed in asolvent, for example an aqueous buffer solution such as acetate orphosphate, at around neutral pH, for example around pH 4.5 to around pH8.5, typically pH 4.5 to 8, suitably pH6 to 7. The reactions maygenerally be performed at any suitable temperature, for example betweenabout 5° C. and about 70° C., for example at room temperature. Thesolvent may optionally contain a chelating agent such as EDTA, EGTA,CDTA or DTPA. Preferably the solvent contains EDTA at between 1 and 5mM, preferably 2 mM. Alternatively or in addition the solvent may be achelating buffer such as citric acid, oxalic acid, folic acid, bicine,tricine, tris or ADA. The PEG will generally be employed in excessconcentration relative to the concentration of the antibody fragment.Typically the PEG is in between 2 and 100 fold molar excess, preferably5, 10 or 50 fold excess.

Where necessary, the desired product containing the desired number ofPEG molecules may be separated from any starting materials or otherproduct generated during the production process by conventional means,for example by chromatography techniques such as ion exchange, sizeexclusion, protein A, G or L affinity chromatography or hydrophobicinteraction chromatography. To identify inhibitors of IL-17 activity anumber of different approaches may be taken by those skilled in the art.In one example inhibitors are identified by first identifying agentsthat interact with IL-17 or IL-17R and subsequently testing those agentsto identify those that inhibit IL-17 activity. In one such example theagent is an antibody.

Agents that interact with IL-17 or IL-17R may be identified using anysuitable method, for example by using a cell-free or cell-based assaysystem where the IL-17 or IL-17R polypeptide is contacted with acandidate agent and the ability of the candidate agent to interact withthe polypeptide is determined. Preferably, the ability of a candidateagent to interact with a IL-17 or IL-17R polypeptide is compared to areference range or control. If desired, this assay may be used to screena plurality (e.g. a library) of candidate agents using a plurality ofIL-17 or IL-17R polypeptide samples. In one example of a cell freeassay, a first and second sample comprising native or recombinant IL-17or IL-17R polypeptide are contacted with a candidate agent or a controlagent and the ability of the candidate agent to interact with thepolypeptide is determined by comparing the difference in interactionbetween the candidate agent and control agent. Preferably, thepolypeptide is first immobilized, by, for example, contacting thepolypeptide with an immobilized antibody which specifically recognizesand binds it, or by contacting a purified preparation of polypeptidewith a surface designed to bind proteins. The polypeptide may bepartially or completely purified (e.g. partially or completely free ofother polypeptides) or part of a cell lysate. Further, the polypeptidemay be a fusion protein comprising the IL-17 or IL-17R polypeptide or abiologically active portion thereof and a domain such asglutathionine-S-transferase or the Fc region of IgG1. Alternatively, thepolypeptide can be biotinylated using techniques well known to those ofskill in the art (e.g. biotinylation kit, Pierce Chemicals; Rockford,Ill.). The ability of the candidate agent to interact with thepolypeptide can be determined by methods known to those of skill in theart for example, ELISA, BIAcore™, Flow cytometry or fluorescentmicrovolume assay technology (FMAT). In another example where acell-based assay is used, a population of cells expressing IL-17 orIL-17R is contacted with a candidate agent and the ability of thecandidate agent to interact with the polypeptide is determined.Preferably, the ability of a candidate agent to interact with IL-17 orIL-17R is compared to a reference range or control. The cell, forexample, can be of eukaryotic origin (e.g. yeast or mammalian) and canexpress the IL-17 or IL-17R polypeptide endogenously or be geneticallyengineered to express the polypeptide. In some instances, the IL-17 orIL-17R polypeptide or the candidate agent is labelled, for example witha radioactive label (such as ³²P, ³⁵S or ¹²⁵I) or a fluorescent label(such as fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enabledetection of an interaction between a polypeptide and a candidate agent.Alternative methods such as ELISA, flow cytometry and FMAT may also beused.

Agents which inhibit IL-17 activity may be identified by any suitablemethod, for example by:

-   -   (i) comparing the activity of IL-17 in the presence of a        candidate agent with the activity of said polypeptide in the        absence of the candidate agent or in the presence of a control        agent; and    -   (ii) determining whether the candidate agent inhibits activity        of IL-17.

Such assays can be used to screen candidate agents, in clinicalmonitoring or in drug development.

As described above, agents may be pre-screened where appropriate toidentify agents (e.g. an antibody) that interact with IL-17 or IL-17Rprior to screening those agents which bind for their ability to inhibitIL-17 activity.

In one example a cell-based assay system is used to identify agentscapable of inhibiting the activity of IL-17. In one particular examplethe assay used to identify inhibitors of IL-17 activity is the standardIL-6 release assay from fibroblasts (Yao et al., 1995, Journal ofImmunology, 155,5483-5486). Potential inhibitors are added to the assayand IL-6 release determined by ELISA. Inhibition is therefore measuredas a reduction in IL-6 release compared to controls.

In another example inhibitors of IL-17 may down-regulate the expressionof the IL-17 or IL-17R polypeptide, for example antisense inhibitors.Such inhibitors may be identified by any method known in the art. In oneexample such inhibitors are identified in a cell-based assay system.Accordingly, a population of cells expressing a IL-17 or IL-17Rpolypeptide or nucleic acid are contacted with a candidate agent and theability of the candidate agent to alter expression of the IL-17 orIL-17R polypeptide or nucleic acid is determined by comparison to areference range or control. In one example, populations of cellsexpressing a IL-17 or IL-17R polypeptide are contacted with a candidateagent or a control agent and the ability of the candidate agent to alterthe expression of the IL-17 or IL-17R polypeptides or nucleic acids isdetermined by comparing the difference in the level of expression of theIL-17 or IL-17R polypeptides or nucleic acids between the treated andcontrol populations of cells. If desired, this assay may be used toscreen a plurality (e.g. a library) of candidate agents. The cell, forexample, can be of eukaryotic origin (e.g. yeast or mammalian) and canexpress an IL-17 or IL-17R polypeptide endogenously or be geneticallyengineered to express a IL-17 or IL-17R polypeptide. The ability of thecandidate agents to alter the expression of a said polypeptides ornucleic acids can be determined by methods known to those of skill inthe art, for example and without limitation, by flow cytometry,radiolabelling, a scintillation assay, immunoprecipitation, Western blotanalysis, Northern blot analysis or RT-PCR.

Agents that inhibit the activity of IL-17 may be identified or furthertested, for example to determine therapeutically effective amounts inone or more animal models. Examples of suitable animals include, but arenot limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs andcats. Preferably, the animal used represents a model of MS.

In one example where the agent inhibits the expression of IL-17 orIL-17R, a first and second group of mammals are administered with acandidate agent or a control agent and the ability of the candidateagent to inhibit the expression of IL-17 or IL-17R polypeptide ornucleic acid is determined by comparing the difference in the level ofexpression between the first and second group of mammals. Where desired,the expression levels of the IL-17 or IL-17R polypeptides or nucleicacid in the first and second groups of mammals can be compared to thelevel of IL-17 or IL-17R polypeptide or nucleic acid in a control groupof mammals. The candidate agent or a control agent can be administeredby means known in the art (e.g. orally, rectally or parenterally such asintraperitoneally or intravenously). Changes in the expression of apolypeptide or nucleic acid can be assessed by the methods outlinedabove.

In another example, the inhibition of IL-17 activity can be determinedby monitoring an amelioration or improvement in disease symptoms, adelayed onset or slow progression of the disease, for example butwithout limitation, a reduction in paralysis. Techniques known tophysicians familiar with MS can be used to determine whether a candidateagent has altered one or more symptoms associated with the disease.

A number of different models of MS are known in the art ('t Hart andAmor 2003, Current Opinion in Neurology 16:375-83). In particular,experimental autoimmune encephalomyelitis (EAE) in ABH mice isconsidered to be a relevant model for MS in humans (Baker et al., 1990.Journal of Neuroimmunology, 28:261-270). Both acute andrelapsing-remitting models have been developed.

As discussed herein, inhibitors of IL-17 activity can be used in thetreatment and/or prophylaxis of MS. For such use the agents willgenerally be administered in the form of a pharmaceutical composition.

Also provided is a pharmaceutical composition comprising an inhibitor ofIL-17 activity and a pharmaceutically acceptable carrier.

The term ‘treatment’ includes either therapeutic or prophylactictherapy. When a reference is made herein to a method of treating orpreventing a disease or condition using a particular inhibitor orcombination of inhibitors, it is to be understood that such a referenceis intended to include the use of that inhibitor or combination ofinhibitors for the manufacture of a medicament for the treatment and/orprophylaxis of MS.

The composition will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. This composition may be in any suitable form(depending upon the desired method of administering it to a patient).

The inhibitors of use in the invention are preferably administered to asubject by a variety of other routes such as orally, transdermally,subcutaneously, intranasally, intravenously, intramuscularly,intrathecally and intracerebroventricularly. The most suitable route foradministration in any given case will depend on the particularinhibitor, the subject, and the nature and severity of the disease andthe physical condition of the subject.

The inhibitors of use in the invention may be administered incombination, e.g. simultaneously, sequentially or separately, with oneor more other therapeutically active compounds, which may be for exampleother anti-MS therapies or anti-cancer therapies.

Pharmaceutical compositions may be conveniently presented in unit doseforms containing a predetermined amount of an active agent of theinvention per dose. Such a unit may contain for example but withoutlimitation, 750 mg/kg to 0.1 mg/kg depending on the condition beingtreated, the route of administration and the age, weight and conditionof the subject.

Pharmaceutically acceptable carriers for use in the invention may take awide variety of forms depending, e.g. on the route of administration.

Compositions for oral administration may be liquid or solid. Oral liquidpreparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Oral liquid preparations may containsuspending agents as known in the art.

In the case of oral solid preparations such as powders, capsules andtablets, carriers such as starches, sugars, microcrystalline cellulosegranulating agents, lubricants, binders, disintegrating agents, and thelike may be included. Because of their ease of administration, tabletsand capsules represent the most advantageous oral dosage unit form inwhich case solid pharmaceutical carriers are generally employed. Inaddition to the common dosage forms set out above, active agents of theinvention may also be administered by controlled release means and/ordelivery devices. Tablets and capsules may comprise conventionalcarriers or excipients such as binding agents for example, syrup,acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers,for example lactose, sugar, maize-starch, calcium phosphate, sorbitol orglycine; tableting lubricants, for example magnesium stearate, talc,polyethylene glycol or silica; disintegrants, for example potato starch;or acceptable wetting agents such as sodium lauryl sulphate. The tabletsmay be coated by standard aqueous or non-aqueous techniques according tomethods well known in normal pharmaceutical practice.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeagent, as a powder or granules, or as a solution or a suspension in anaqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or awater-in-oil liquid emulsion. Such compositions may be prepared by anyof the methods of pharmacy but all methods include the step of bringinginto association the active agent with the carrier, which constitutesone or more necessary ingredients. In general, the compositions areprepared by uniformly and intimately admixing the active agent withliquid carriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired presentation. Forexample, a tablet may be prepared by compression or moulding, optionallywith one or more accessory ingredients.

Pharmaceutical compositions suitable for parenteral administration maybe prepared as solutions or suspensions of the active agents of theinvention in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include aqueous ornon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the composition isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. Extemporaneous injection solutions, dispersions and suspensionsmay be prepared from sterile powders, granules and tablets.

Pharmaceutical compositions can be administered with medical devicesknown in the art. For example, in a preferred embodiment, apharmaceutical composition of the invention can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;4,790,824; or 4,596,556. Examples of well-known implants and modulesuseful in the present invention include: U.S. Pat. No. 4,487,603, whichdiscloses an implantable micro-infusion pump for dispensing medicationat a controlled rate; U.S. Pat. No. 4,486,194, which discloses atherapeutic device for administering medicaments through the skin; U.S.Pat. No. 4,447,233, which discloses a medication infusion pump fordelivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which, discloses a variable flow implantable infusionapparatus for continuous drug delivery, U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known to those skilled in the art.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, impregnated dressings, sprays, aerosols oroils, transdermal devices, dusting powders, and the like. Thesecompositions may be prepared via conventional methods containing theactive agent. Thus, they may also comprise compatible conventionalcarriers and additives, such as preservatives, solvents to assist drugpenetration, emollients in creams or ointments and ethanol or oleylalcohol for lotions. Such carriers maybe present as from about 1% up toabout 98% of the composition. More usually they will form up to about80% of the composition. As an illustration only, a cream or ointment isprepared by mixing sufficient quantities of hydrophilic material andwater, containing from about 5-10% by weight of the compound, insufficient quantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active agent may be delivered from the patch byiontophoresis.

For applications to external tissues, for example the mouth and skin,the compositions are preferably applied as a topical ointment or creamWhen formulated in an ointment, the active agent may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active agent may be formulated in a cream with an oil-in-water creambase or a water-in-oil base.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for topical administration to theeye include eye drops wherein the active agent is dissolved or suspendedin a suitable carrier, especially an aqueous solvent. They also includetopical ointments or creams as above.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter or other glycerideor materials commonly used in the art, and the suppositories may beconveniently formed by admixture of the combination with the softened ormelted carrier(s) followed by chilling and shaping moulds. They may alsobe administered as enemas.

The dosage to be administered of an inhibitor of IL-17 activity willvary according to the particular inhibitor, the type of MS, the subject,and the nature and severity of the disease and the physical condition ofthe subject, and the selected route of administration; the appropriatedosage can be readily determined by a person skilled in the art. For thetreatment and/or prophylaxis of MS in humans and animals pharmaceuticalcompositions comprising antibodies can be administered to patients(e.g., human subjects) at therapeutically or prophylactically effectivedosages (e.g. dosages which result in inhibition of MS and/or relief ofMS symptoms) using any suitable route of administration, such asinjection and other routes of administration known in the art forclinical products, such as antibody-based clinical products.

The compositions may contain from 0.1% by weight, preferably from10-60%, or more, by weight, of the inhibitor of the invention, dependingon the method of administration.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an inhibitor of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and the ageand condition of the particular subject being treated, and that aphysician will ultimately determine appropriate dosages to be used. Thisdosage may be repeated as often as appropriate. If side effects developthe amount and/or frequency of the dosage can be altered or reduced, inaccordance with normal clinical practice.

In another example, where the inhibitor is a nucleic acid this may beadministered via gene therapy (see for example Hoshida, T. et al., 2002,Pancreas, 25:111-121; Ikuno, Y. 2002, Invest. Ophthalmol. Vis. Sci. 200243:2406-2411; Bollard, C., 2002, Blood 99:3179-3187; Lee E., 2001, Mol.Med. 7:773-782). Gene therapy refers to administration to a subject ofan expressed or expressible nucleic acid. In one example this is eitherthe IL-17 or the IL-17R nucleic acid or portions thereof. Any of themethods for gene therapy available in the art can be used according tothe present invention.

Delivery of the therapeutic nucleic acid into a patient can be direct invivo gene therapy (i.e. the patient is directly exposed to the nucleicacid or nucleic acid-containing vector) or indirect ex vivo gene therapy(i.e. cells are first transformed with the nucleic acid in vitro andthen transplanted into the patient).

For example for in vivo gene therapy, an expression vector containingthe IL-17 or IL-17R nucleic acid may be administered in such a mannerthat it becomes intracellular, i.e. by infection using a defective orattenuated retroviral or other viral vectors as described, for example,in U.S. Pat. No. 4,980,286 or by Robbins et al., 1998, Pharmacol. Ther.80:35-47.

The various retroviral vectors that are known in the art are such asthose described in Miller et al. (1993, Meth. Enzymol. 217:581-599)which have been modified to delete those retroviral sequences which arenot required for packaging of the viral genome and subsequentintegration into host cell DNA. Also adenoviral vectors can be usedwhich are advantageous due to their ability to infect non-dividing cellsand such high-capacity adenoviral vectors are described in Kochanek(1999, Human Gene Therapy, 10:2451-2459). Chimeric viral vectors thatcan be used are those described by Reynolds et al. (1999, MolecularMedicine Today, 1:25-31). Hybrid vectors can also be used and aredescribed by Jacoby et al. (1997, Gene Therapy, 4:1282-1283).

Direct injection of naked DNA or through the use of microparticlebombardment (e.g. Gene Gun®; Biolistic, Dupont) or by coating it withlipids can also be used in gene therapy. Cell-surfacereceptors/transfecting compounds or through encapsulation in liposomes,microparticles or microcapsules or by administering the nucleic acid inlinkage to a peptide which is known to enter the nucleus or byadministering it in linkage to a ligand predisposed to receptor-mediatedendocytosis (See Wu & Wu. 1987, J. Biol. Chem., 262:4429-4432) can beused to target cell types which specifically express the receptors ofinterest.

In ex vivo gene therapy, a gene is transferred into cells in vitro usingtissue culture and the cells are delivered to the patient by variousmethods such as injecting subcutaneously, application of the cells intoa skin graft and the intravenous injection of recombinant blood cellssuch as haematopoietic stem or progenitor cells.

Cells into which a IL-17 or IL-17R nucleic acid can be introduced forthe purposes of gene therapy include, for example, epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells. The blood cells that can be used include, for example,T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryotcytes, granulocytes, haematopoietic cells orprogenitor cells, and the like.

In a one aspect, the pharmaceutical composition of the present inventioncomprises an IL-17 or IL-17R nucleic acid, said nucleic acid being partof an expression vector that expresses an IL-17 or IL-17R polypeptide orchimeric protein thereof in a suitable host. In particular, such anucleic acid has a promoter operably linked to the polypeptide codingregion, said promoter being inducible or constitutive (and, optionally,tissue-specific).

The invention will now be described with reference to the followingexamples, which are merely illustrative and should not in any way beconstrued as limiting the scope of the present invention.

FIGURES

FIG. 1. Effect of Ab#13 mIgG1 on clinical disease when dosed from day—1through the acute phase (black arrowheads, days—1, 6, 13, 20). Averageclinical score (±sd) plotted against day of disease induction (a) andaverage change in weight from day 0 (b).

FIG. 2. Analysis of the acute phase of disease following dosing ofAb#13mIgG1 through the acute phase.

FIG. 3. Analysis of the relapse phase of disease following dosing ofAb#13mIgG1 through the acute phase.

FIG. 4. Effect of Ab#13mIgG1 on clinical disease when dosed through therelapse phase (days 28, 35, 42 and 49, black arrowheads). Averageclinical score (±sd) versus day of disease induction (a) and averagechange in weight from day 0 (b).

FIG. 5. Analysis of the acute phase of disease following dosing ofAb#13mIgG1 through the reipase phase.

FIG. 6. Analysis of the relapse phase of disease following dosing ofAb#13mIgG1 through the relapse phase.

FIG. 7. Effect of Ab#13 Fab-di-PEG and Ab#13 mIgG1 on clinical diseasewhen dosed through the relapse phase (black arrowheads represent dosingdays). Average clinical score (±sd) plotted against day of diseaseinduction (a) and average change in weight from day 0 (b).

FIG. 8. Analysis of acute phase prior to dosing with Ab#13 Fab-di-PEGand Ab#13 mIgG1.

FIG. 9. Analysis of relapse phase following dosing with Ab#13 Fab-di-PEGand Ab#13 mIgG1 during remission.

FIG. 10. Effect of Ab#13mIgG1 on clinical disease when dosed from day-1(black arrowheads represent dosing days, prophylactically (a) andtherapeutically (b).

FIG. 11. Analysis of prohylactic dosing regime.

FIG. 12. Analysis of therapeutic dosing regime.

FIG. 13. Human and murine constant regions.

EXAMPLES Example 1 Isolation of an Anti-IL-17 Antibody

Rabbits were immunised three times with human IL-17 and then twice withmouse IL-1 7. Using a haemolytic plaque assay with biotinylated sheepred blood cells coated with murine IL-17 via streptavidin, 9 antibodygenes were isolated using the methods described by Babcook et al., 1996,Proc. Natl. Acad. Sci, 93,7843-7848 and in WO92/02551. The antibodygenes were expressed in CHO cells and the recombinant antibodiesscreened for their ability to neutralise murine IL-17 in a bioassayusing mouse 3T3-NIH cells (Yao et al., 1995, Immunity, 3:811-821). Allthe antibodies in the panel neutralised murine IL-17 in this assay andone antibody, m170013 (Ab#13) was selected for in vivo testing. Fortesting the efficacy of the antibody in EAE, a chimeric IgG (Ab#13mIgG1) was produced using the rabbit variable region from antibodym170013 and mouse constant regions.

Example 2 Effect of Ab#13mIgG1on the Symptoms of EAE

The MS model, experimental autoimmune encephalomyelitis (EAE), was usedessentially as described by Baker et al., 1990. Journal ofNeuroimmunology, 28:261-270. Female ABH mice 8-10 weeks of age (Harlan)were immunised with mouse spinal cord homogenate (SCH, 3.33 mg/ml) incomplete freund's adjuvant by subcutaneous immunisation in either flank(150 μl/site) on days 0 and 7.

i) Dosing Over the Acute Phase

Two groups were dosed with antibody at 10 mg/kg, sc on days—1, 6, 13 and20. One group (n=14) was dosed with Ab#13 mIgG1 the other (n=13) with101.4 (isotype control).

ii) Dosing Over the Relapsing Phase

A total of 30 mice were followed through the acute phase of disease andon day 27 analysis of the acute phase of disease was performed to selecttwo groups with similar disease profiles in the acute phase (day ofonset, peak disease score, cumulative clinical score and weight loss).Two groups of 12 mice were selected for dosing with antibody at 10mg/kg, sc on days 28, 35, 42 and 49. One group was dosed with Ab#13mIgG1) the other with 101.4 (isotype control).

Weights and clinical scores were recorded daily by an assessor blindedto treatment and terminal EDTA-Plasma collected. Clinical score scale 0Normal 0.25 Tail dragging 0.5 Partial tail paralysis 1 Complete tailparalysis 2 Incomplete hind limb paralysis 3 Complete hindparalysis/incontinence 4 Front limb paralysis/loss of righting reflex

Statistics

Pairwise comparisons of clinical scores and day of onset were performedusing Mann-Whitney U test, analysis of incidence was performed withFishers exact test, analysis of maximum weight loss was performed usingStudents' T test.

Results

i) Dosing during acute phase: A significant delay in the onset of theacute phase and a reduced severity and incidence of first relapse wasobserved (FIGS. 1, 2 and 3). FIG. 2 shows that Ab#13 mIgG1dosed throughthe acute phase had no effect on the incidence of disease in the acutephase (Ab#13 mIgG1, 13/14 with disease vs 13/13 for isotype control,101.4). The only statistically significant effect was a delay in theonset of the acute phase of disease FIG. 2 a, p=0.0039, Mann-Whitney Utest. No effects were seen on weight loss (b), maximum clinical score(c) or cumulative clinical score (d) in the acute phase. FIG. 3 showsthat Ab#13 mIgG1dosed through the acute phase caused a significantreduction in the incidence of the relapse phase of disease (Ab#13 mIgG1,6/14 with disease vs 11/11 for isotype control, 101.4, p=0.0029, Fishersexact test). There was no statistically significant delay in the onsetof the relapse phase of disease for those animals which entered relapse(a). A significant reduction in weight loss (b, p=0.0001, Student's Ttest), maximum clinical score (c, p=0.0028, Mann-Whitney U test) ) andcumulative clinical score (d, p=0.0001, Mann-Whitney U test) wereobserved during the relapse phase.

ii) Dosing through the relapsing phase: A reduced incidence, delayedonset and reduced severity of the relapse phase was observed (see FIGS.4, 5 and 6). FIG. 5 shows that the dose groups selected to have asimilar acute phase profile, showed no significant differences in any ofthe parameters analysed. FIG. 6 shows that Ab#13 mIgG1dosed through therelapse phase caused a significant reduction in the incidence of therelapse phase of disease (Ab#13 mIgG1, 5/12 with disease vs 12/12 forisotype control, 101.4, p=0.0046, Fishers exact test). There was also astatistically significant delay in the onset of the relapse phase ofdisease for those animals which entered relapse (a, p=0.0061). Asignificant reduction in weight loss (b, p<0.000l, Student's T test),maximum clinical score (c, p=0.001 1, Mann-Whitney U test)) andcumulative clinical score (d, p=0.0023, Mann-Whitney U test) were alsoobserved during the relapse phase.

Summary

Ab#13 mIgG1 antibody was dosed in separate experiments over the acutephase of disease (prophylactic dosing) and over the relapse phase(therapeutic dosing). Effects were most pronounced on relapse with asignificant reduction in the incidence and severity of the relapse phasefor both dosing regimes.

Example 3 Effect of Ab#13 Fab-Di-PEG on the Symptoms of EAE

A Fab fragment, termed Ab#13 Fab-Di-PEG was produced essentially asdescribed in International Patent Application PCT/GB2004/002810 (filedon 1 Jul. 2004). The Fab consisted of the rabbit variable regions ofantibody 13 from example 1 and mouse IgG1 constant regions. In contrastto other Fab fragments, the heavy chain constant region of this Fabterminates at the interchain cysteine of C_(H)1. PCR primers weredesigned based on the murine IgG1 CH1 region and PCR mutagenesis used toinsert a stop codon immediately following the interchain cysteine ofC_(H)1. The mouse constant regions are shown in FIG. 13 and in SEQ IDNos 3 (heavy chain) and 4 (light chain). PCR mutagenesis was also usedto replace the cysteine at position 80 of the rabbit light chainvariable region with alanine. Murine CH1 (SEQ ID NO:3)KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD C* Murine Kappa (SEQID NO:4) DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRGEC*

The Fab fragments were produced in E. coli strain W3110 and purifiedusing standard methods (Humphreys et al., 2002, Protein Expression andPurification, 26, 309-320).

2×20 kDa PEG was attached to the Fab fragment by attaching a linear 20kDa PEG to each of the interchain cysteines (underlined in the sequencesabove). Reductions and PEGylations were performed in 50 mM Tris.HCl 5 mMEDTA pH 7.14 with Fab at 20.06 mg/ml. The Fab was reduced at roomtemperature (˜24° C.) for 30 minutes using 10 mMtris(2-carboxyethyl)phosphine (TCEP) (final). The Fab was desalted on aPD-10 column (Pharmacia) and then mixed with 4 fold molar excess oflinear 20 kDa PEG-maleimide over Fab. The 20 kDa PEG was from NipponOils and Fats (NOF). PEGylated Fab was separated from unPEGylated Fab bysize exclusion HPLC on analytical Zorbax GF-450 and GF-250 columns inseries. These were developed with a 30 min isocratic gradient of 0.2Mphosphate pH 7.0+10% ethanol at 1 ml/min and Fab detected usingabsorbance at 214 nm and 280 nm.

The MS model, experimental autoimmune encephalomyelitis (EAE), was usedessentially as described by Baker et al., 1990. Journal ofNeuroimmunology, 28:261-270.

Female ABH mice 8-10 weeks of age (Harlan) were immunised with mousespinal cord homogenate (SCH, 3.33 mg/ml) in complete Freund's adjuvantin two sites, sc, on the flanks (150 μl/site) on days 0 and 7.

Four groups were dosed prior to onset of first relapse (duringremission). Groups were assigned as follows

Group 1 (n=11) anti-mouse IL-17 Ab#13 Fab-di-PEG (100 mg/kg, s.c,weekly)

Group 2 (n=11) anti-mouse IL-17 Ab#13 Fab-di-PEG (30 mg/kg, s.c, weekly)

Group 3 (n=9) anti-mouse IL-17 Ab#13 mIgG1 (10 mg/kg, sc)

Group 4 (n=9) PBS control

Weights and clinical scores were recorded daily and terminal EDTA-Plasmacollected. Clinical score scale 0 Normal 0.25 Tail dragging 0.5 Partialtail paralysis 1 Complete tail paralysis 2 Incomplete hind limbparalysis/loss of righting reflex 3 Complete hind paralysis/incontinence4 Front limb paralysis/moribund

Statistics

Pair wise comparisons of maximum and cumulative clinical scores and dayof onset were performed using Mann-Whitney U test. Cumulative clinicalscore is defined as the sum of clinical scores throughout the diseasecourse for each animal (area under the curve). Comparisons of diseaseincidence were performed using Fishers Exact Test. Maximum weight losswas analysed using one-way Anova with Bonferroni post test.

Results

FIG. 7 a shows the effect of anti-IL17 Ab#13 di Fab-PEG and Ab#13 mIgG1on clinical disease when dosed from remission (black arrowheadsrepresent dosing days). When dosed prior to first relapse all activedoses showed a significant reduction in cumulative and maximum clinicalscore and incidence.

FIG. 8 shows that during the acute phase, prior to antibody treatment,all assigned groups showed no significant differences in disease onsetor clinical severity prior to antibody dosing.

FIG. 9 shows that anti-mouse IL-17 antibodies dosed prior to relapseonset during remission showed a significant reduction in maximumclinical score (Ab#13 di Fab-PEG 100 mg/kg vs. PBS p<0.05 and IL-17Ab#13 mIgG1 vs. PBS p<0.001),

There was also a reduction in cumulative score (Ab#13 di Fab-PEG (100mg/kg and 30 mg/kg) and Ab#13 mIgG1 vs. PBS p<0.01, p<0.05 and p<0.001respectively). Furthermore there was a reduction in maximum weight loss(Ab#13 di Fab-PEG 100 mg/kg and Ab#13 mIgG1 vs. PBS both p<0.05).

Actual incidence of relapse is summarised in table 1, with all activelytreated groups having significantly lower incidence than the PBS controlgroup. TABLE 1 Ab#13 Ab#13 Ab#13 di Fab-PEG di Fab-PEG IgG1 100 mg/kg***30 mg/kg* 10 mg/kg*** PBS Animals 0 2 0 7 entering relapse Animals not11 9 11 2 entering relapse Total number 11 11 11 9 of animals***P = 0.005*p = 0.0216***P = 0.005

Summary

Anti-mouse IL17 antibodies (Ab#13 di Fab-PEG and Ab#13 mIgG1) were dosedin a dose dependent manner during the remission phase prior to onset offirst relapse. Effects were pronounced with a significant reduction forboth antibodies in relapse incidence and upon maximum and cumulativedisease score in comparison to the PBS control group.

Example 4 Effect of Ab#13 mIgG1 on the Symptoms of Chronic EAE inC57BI/6 Mice

The chronic EAE model used was essentially as described by Copray etal., 2004. Journal of Neuroimmunology, 148:41-53.

Female C57B1/6 mice 6-8 weeks of age (Charles River) were immunised withMyelin Oligodendrocyte Protein (MOG 35-55, 0.66 mg/ml) in completeFreund's adjuvant (0.4 mg/ml Mycobacterium; 4:1 M.tuberculosis: Mbutyricum) in two sites, s.c, on the flanks (150 μl/site) on days 0 and7. Mice were also administered pertussis toxin (1 μg/ml) on days0,1,7,8; 200 μl i.p

Two groups were dosed prophylactically (10 mg/kg; s.c; commencing onPSD—1 until the end of experiment). One group (n=15) was dosed withAb#13 mIgG1, (chimeric rabbit V region, Murine constant region IgG-1, asdescribed in Example 1), the other (n=15) with 101.4 (Murine IgG-1isotype control, 101.4).

Two groups were dosed therapeutically (upon 50% incidence) with Ab#13mIgG1 and control antibody 101.4 (10 mg/kg; s.c commencing PSD 16 tillend of experiment) Weights and clinical scores were recorded daily andterminal EDTA-Plasma collected. Clinical score scale 0 Normal 0.25 Taildragging 0.5 Partial tail paralysis 1 Complete tail paralysis 2Incomplete hind limb paralysis/loss of righting reflex 3 Complete hindparalysis/incontinence 4 Front limb paralysis/moribund

Statistics

Statistical analysis was performed as described in Example 3.

Results

FIG. 10 shows that prophylactic dosing (a) of Ab#13 mIgG1 elicitedsignificant delay in the onset of disease, cumulative and maximumclinical score. Therapeutic dosing (b) significantly reduced cumulativeclinical score. Further analysis showed Ab#13 mIgG1 dosedprophylactically had significant effects in reducing cumulative score(p=0.0006), delay of onset (p=0.0184) and maximum clinical score(p=0.0032, all Mann-Whitney U test) (FIG. 11). Ab#13 mIgG1 dosedtherapeutically also showed a significant reduction in cumulative score(b, p=0.0229, Mann-Whitney U test; FIG. 12).

Summary

Anti-mouse IL17 antibody Ab#13 mIgG1 was dosed in separate experimentsprophylactically and therapeutically. Effects were most pronouncedprophylactically with a significant reduction in maximum and cumulativedisease score, furthermore incidence of disease was significantlydelayed. Therapeutic treatment showed a reduction in cumulative diseasescore.

1-9. (canceled)
 10. A method for the treatment or prophylaxis ofmultiple sclerosis (MS) comprising administering a therapeuticallyeffective amount of an inhibitor of IL-17 activity to a patient in needthereof.
 11. The method according to claim 10, wherein the inhibitor isa small molecule.
 12. The method according to claim 10, wherein theinhibitor is a nucleic acid.
 13. The method according to claim 10,wherein the inhibitor is an antibody or a functionally active antibodyfragment or derivative.
 14. The method according to claim 13, whereinthe antibody or antibody fragment is monoclonal, polyclonal, chimeric,humanized or bispecific.
 15. The method according to claim 13 whereinthe antibody fragment is a Fab, Fab′, F(ab′)₂, scFv or epitope bindingfragment.
 16. The method according to claim 13 wherein the antibody orantibody fragment is conjugated to one or more effector molecule(s). 17.The method according to claim 13 wherein the antibody or antibodyfragment binds to IL-17.
 18. The method according to claim 13 whereinthe antibody or antibody fragment binds to IL-17R.
 19. The methodaccording to claim 10 wherein the inhibitor of IL-17 activity isadministered in combination with one or more other therapeuticallyactive compounds.